Enzymes for starch processing

ABSTRACT

The present invention relates to a hybrid enzyme comprising carbohydrate-binding module amino acid sequence and a fungal alpha-amylase amino acid sequence and to a variant of a fungal wild type enzyme comprising a carbohydrate-binding module and an alpha-amylase catalytic module. The invention also relates to the use of the hybrid enzyme or the variant in starch liquefaction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/877,849 filed on Jun. 25, 2004, which claims the benefit or priorityof Danish application nos. PA 2003 00949 and PA 2003 01568 filed Jun.25, 2003 and Oct. 24, 2003, respectively, and U.S. provisionalapplication Nos. 60/482,589, 60/490,751, 60/511,044, 60/514,854, and60/569,862 filed Jun. 25, 2003, Jul. 29, 2003, Oct. 14, 2003, Oct. 27,2003, and May 10, 2004, respectively, the contents of which are fullyincorporated herein by reference.

SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates, inter alia, to an enzyme comprising acarbohydrate-binding module (“CBM”) and an alpha-amylase catalyticdomain. The enzyme may be a hybrid between a carbohydrate-binding module(“CBM”) and an alpha-amylase or the enzyme may be a variant of a parentenzyme comprising a carbohydrate-binding module (“CBM”) and analpha-amylase catalytic domain. The invention also relates to the use ofthe enzyme in a starch liquefaction process in which starch is degradedto smaller oligo- and/or polysaccharide fragments.

BACKGROUND OF THE INVENTION

A large number of enzymes and processes have been described forconverting starch to starch hydrolysates, such as maltose, glucose orspecialty syrups, either for use as sweeteners or as precursors forother saccharides such as fructose. Glucose may also be fermented toethanol or other fermentation products, such as citric acid, monosodiumglutamate, gluconic acid, sodium gluconate, calcium gluconate, potassiumgluconate, glucono delta lactone, or sodium erythorbate, itaconic acid,lactic acid, gluconic acid; ketones; amino acids, glutamic acid (sodiummonoglutaminate), penicillin, tetracyclin; enzymes; vitamins, such asriboflavin, B12, beta-carotene or hormones.

Starch is a high molecular-weight polymer consisting of chains ofglucose units. It usually consists of about 80% amylopectin and 20%amylose. Amylopectin is a branched polysaccharide in which linear chainsof alpha-1,4 D-glucose residues are joined by alpha-1,6 glucosidiclinkages.

Amylose is a linear polysaccharide built up of D-glucopyranose unitslinked together by alpha-1,4 glucosidic linkages. In the case ofconverting starch into a soluble starch hydrolysate, the starch isdepolymerized. The conventional depolymerization process consists of agelatinization step and two consecutive process steps, namely aliquefaction process and a saccharification process.

Granular starch consists of microscopic granules, which are insoluble inwater at room temperature. When an aqueous starch slurry is heated, thegranules swell and eventually burst, dispersing the starch moleculesinto the solution. During this “gelatinization” process there is adramatic increase in viscosity. As the solids level is 30-40% in atypical industrial process, the starch has to be thinned or “liquefied”so that it can be handled. This reduction in viscosity is today mostlyobtained by enzymatic degradation. During the liquefaction step, thelong-chained starch is degraded into smaller branched and linear units(maltodextrins) by an alpha-amylase. The liquefaction process istypically carried out at about 105-110° C. for about 5 to 10 minutesfollowed by about 1-2 hours at about 95° C. The temperature is thenlowered to 60° C., a glucoamylase or a beta-amylase and optionally adebranching enzyme, such as an isoamylase or a pullulanase are added,and the saccharification process proceeds for about 24 to 72 hours.

It will be apparent from the above discussion that the conventionalstarch conversion process is very energy consuming due to the differentrequirements in terms of temperature during the various steps. It isthus desirable to be able to select and/or design the enzymes used inthe process so that the overall process can be performed without havingto gelatinize the starch. Such processes are the subject of U.S. Pat.Nos. 4,591,560, 4,727,026 and 4,009,074 and EP 0171218, and Danishapplication no. PA 2003 00949. The present invention discloses a newhybrid enzyme and a genetic modification of a wild type enzyme designedfor such processes and comprising an amino acid sequence of a CBM and anamino acid sequence of a fungal starch degrading enzyme. Hybrid enzymesare the subject of WO 98/14601, WO 00/77165 and Danish application no.PA 2003 00949.

SUMMARY OF THE INVENTION

The invention provides in a first aspect a hybrid enzyme which comprisesan amino acid sequence of a catalytic module having alpha-amylaseactivity and an amino acid sequence of a carbohydrate-binding module,wherein the catalytic module is of fungal origin.

The invention provides in a second aspect a variant of the fungalalpha-amylase shown as SEQ ID NO:41 or variants of an alpha-amylasehaving at least 60% homology, at least 70% homology, at least 80%homology, or even at least 90% homology to SEQ ID NO:41, which variantcomprising an alteration at one or more of the positions: 13, 15, 18,31, 32, 33, 34, 35, 36, 61, 63, 64, 68, 69, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 89, 117, 118, 119, 120, 121, 122, 123, 124, 125,152, 153, 154, 155, 156, 157, 158, 161, 162, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 204, 205, 206, 207, 208, 209, 210, 211,216, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 242, 245,250, 252, 253, 255, 256, 257, 259, 260, 275, 292, 295, 296, 297, 298,299, 304, 328, 339, 344, 348, 378, 383, 386, 387, 405, 448 and 480wherein (a) the alteration(s) are independently, i) an insertion of anamino acid downstream of the amino acid which occupies the position, ii)a deletion of the amino acid which occupies the position, or iii) asubstitution of the amino acid which occupies the position with adifferent amino acid, (b) the variant has increased acid alpha-amylaseactivity and or improved enzyme stability relative to the parent fungalalpha-amylase and, (c) each position corresponds to a position of theamino acid sequence of the TAKA amylase shown in SEQ ID NO:43 and/or theA. kawachii alpha-amylase shown as SEQ ID NO:41.

In further aspects the invention provides an isolated DNA sequenceencoding the hybrid enzyme of the first aspect or the variant of thesecond aspect, a DNA construct comprising the DNA sequence encoding thehybrid enzyme of the first aspect or the variant of the second aspect,an expression vector comprising the DNA sequence encoding the hybridenzyme of the first aspect or the variant of the second aspect, and ahost cell transformed with a vector; which host cell is capable ofexpressing the DNA sequence encoding the hybrid enzyme of the firstaspect or the variant of the second aspect.

In an eighth aspect the invention provides a method for liquefyingstarch, wherein a gelatinized or granular starch substrate is treated inaqueous medium with the hybrid enzyme of the first aspect or the variantof the second aspect.

DETAILED DESCRIPTION OF THE INVENTION

The term “granular starch” is understood as raw uncooked starch, i.e.,starch that has not been subjected to a gelatinization. Starch is formedin plants as tiny granules insoluble in water. These granules arepreserved in starches at temperatures below the initial gelatinizationtemperature. When put in cold water, the grains may absorb a smallamount of the liquid. Up to 50° C. to 70° C. the swelling is reversible,the degree of reversibility being dependent upon the particular starch.With higher temperatures an irreversible swelling called gelatinizationbegins.

The term “initial gelatinization temperature” is understood as thelowest temperature at which gelatinization of the starch commences.Starch heated in water begins to gelatinize between 50° C. and 75° C.;the exact temperature of gelatinization depends on the specific starchand can readily be determined by the skilled artisan. Thus, the initialgelatinization temperature may vary according to the plant species, tothe particular variety of the plant species as well as with the growthconditions. In the context of this invention the initial gelatinizationtemperature of a given starch is the temperature at which birefringenceis lost in 5% of the starch granules using the method described byGorinstein and Lii, 1992, Starch/Stärke 44(12): 461-466.

The term “soluble starch hydrolysate” is understood as the solubleproducts of the processes of the invention and may comprise mono-, di-,and oligosaccharides, such as glucose, maltose, maltodextrins,cyclodextrins and any mixture of these. Preferably at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97% or 98% of the dry solids of the granularstarch is converted into a soluble starch hydrolysate.

The term polypeptide “homology” is understood as the degree of identitybetween two sequences indicating a derivation of the first sequence fromthe second. The homology may suitably be determined by means of computerprograms known in the art such as GAP provided in the GCG programpackage (Program Manual for the Wisconsin Package, Version 8, August1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA53711) (Needleman and Wunsch, 1970, Journal of Molecular Biology 48:443-453. The following settings for amino acid sequence comparison areused: GAP creation penalty of 3.0 and GAP extension penalty of 0.1. Therelevant part of the amino acid sequence for the homology determinationis the mature polypeptide, i.e., without the signal peptide.

Hybrid Enzymes

Enzyme classification numbers (EC numbers) referred to in the presentspecification with claims are in accordance with the Recommendations ofthe Nomenclature Committee of the International Union of Biochemistryand Molecular Biology, Academic Press Inc, 1992.

Hybrid enzymes or a genetically modified wild type enzymes as referredto herein include species comprising an amino acid sequence of analpha-amylolytic enzyme (EC 3.2.1.1) linked (i.e., covalently bound) toan amino acid sequence comprising a carbohydrate-binding module (CBM).

CBM-containing hybrid enzymes, as well as detailed descriptions of thepreparation and purification thereof, are known in the art [see, e.g.,WO 90/00609, WO 94/24158 and WO 95/16782, as well as Greenwood et al.,1994, Biotechnology and Bioengineering 44: 1295-1305]. They may, e.g.,be prepared by transforming into a host cell a DNA construct comprisingat least a fragment of DNA encoding the carbohydrate-binding moduleligated, with or without a linker, to a DNA sequence encoding the enzymeof interest, and growing the transformed host cell to express the fusedgene. The resulting recombinant product (hybrid enzyme)—often referredto in the art as a “fusion protein—may be described by the followinggeneral formula:

A-CBM-MR-X

In the latter formula, A-CBM is the N-terminal or the C-terminal regionof an amino acid sequence comprising at least the carbohydrate-bindingmodule (CBM) per se. MR is the middle region (the “linker”), and X isthe sequence of amino acid residues of a polypeptide encoded by a DNAsequence encodng the enzyme (or other protein) to which the CBM is to belinked.

The moiety A may either be absent (such that A-CBM is a CBM per se,i.e., comprises no amino acid residues other than those constituting theCBM) or may be a sequence of one or more amino acid residues(functioning as a terminal extension of the CBM per se). The linker (MR)may be a bond, or a short linking group comprising from about 2 to about100 carbon atoms, in particular of from 2 to 40 carbon atoms. However,MR is preferably a sequence of from about 2 to about 100 amino acidresidues, more preferably of from 2 to 40 amino acid residues, such asfrom 2 to 15 amino acid residues.

The moiety X may constitute either the N-terminal or the C-terminalregion of the overall hybrid enzyme.

It will thus be apparent from the above that the CBM in a hybrid enzymeof the type in question may be positioned C-terminally, N-terminally orinternally in the hybrid enzyme.

Linker Sequence

The linker sequence may be any suitable linker sequence. In preferredembodiments the linker sequence is derived from the Athelia rolfsii AMG,the A. niger AMG or the A. kawachii alpha-amylase such as a linkersequence selected from the list consisting of A. niger AMG linker: T G GT T T T A T P T G S G S V T S T S K T T A T A S K T S T S T S S T S A(SEQ ID NO:26), A. kawachii alpha-amylase linker: T T T T T T A A A T ST S K A T T S S S S S S A A A T T S S S (SEQ ID NO:27), Athelia rolfsiiAMG linker: G A T S P G G S S G S (SEQ ID NO:28), and the PEPT linker: PE P T P E P T (SEQ ID NO:29). In another preferred embodiment the hybridenzyme has a linker sequence which differs from the amino acid sequenceshown in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29 in nomore than 10 positions, no more than 9 positions, no more than 8positions, no more than 7 positions, no more than 6 positions, no morethan 5 positions, no more than 4 positions, no more than 3 positions, nomore than 2 positions, or even no more than 1 position.

Carbohydrate-Binding Modules

A carbohydrate-binding module (CBM), or as often referred to, acarbohydrate-binding domain (CBD), is a polypeptide amino acid sequencewhich binds preferentially to a poly- or oligosaccharide (carbohydrate),frequently—but not necessarily exclusively—to a water-insoluble(including crystalline) form thereof.

CBMs derived from starch degrading enzymes are often referred to asstarch-binding modules or SBMs (CBMs which may occur in certainamylolytic enzymes, such as certain glucoamylases, or in enzymes such ascyclodextrin glucanotransferases, or in alpha-amylases). Likewise, othersub-classes of CBMs would embrace, e.g., cellulose-binding modules (CBMsfrom cellulolytic enzymes), chitin-binding modules (CBMs which typicallyoccur in chitinases), xylan-binding modules (CBMs which typically occurin xylanases), mannan-binding modules (CBMs which typically occur inmannanases). SBMs are often referred to as SBDs (Starch BindingDomains).

CBMs are found as integral parts of large polypeptides or proteinsconsisting of two or more polypeptide amino acid sequence regions,especially in hydrolytic enzymes (hydrolases) which typically comprise acatalytic module containing the active site for substrate hydrolysis anda carbohydrate-binding module (CBM) for binding to the carbohydratesubstrate in question. Such enzymes can comprise more than one catalyticmodule and one, two or three CBMs, and optionally further comprise oneor more polypeptide amino acid sequence regions linking the CBM(s) withthe catalytic module(s), a region of the latter type usually beingdenoted a “linker”. Examples of hydrolytic enzymes comprising a CBM—someof which have already been mentioned above—are cellulases, xylanases,mannanases, arabinofuranosidases, acetylesterases and chitinases. CBMshave also been found in algae, e.g., in the red alga Porphyra purpureain the form of a non-hydrolytic polysaccharide-binding protein.

In proteins/polypeptides in which CBMs occur (e.g., enzymes, typicallyhydrolytic enzymes), a CBM may be located at the N or C terminus or atan internal position.

That part of a polypeptide or protein (e.g., hydrolytic enzyme) whichconstitutes a CBM per se typically consists of more than about 30 andless than about 250 amino acid residues.

The “Carbohydrate-Binding Module of Family 20” or a CBM-20 module is inthe context of this invention defined as a sequence of approximately 100amino acids having at least 45% homology to the Carbohydrate-BindingModule (CBM) of the polypeptide disclosed in FIG. 1 by Joergensen etal., 1997, Biotechnol. Lett. 19: 1027-1031. The CBM comprises the last102 amino acids of the polypeptide, i.e., the subsequence from aminoacid 582 to amino acid 683. The numbering of Glycoside HydrolaseFamilies applied in this disclosure follows the concept of Coutinho andHenrissat, 1999, CAZy—Carbohydrate-Active Enzymes server at URL:afmb.cnrs-mrs.fr/˜cazy/CAZY/index.html or alternatively Coutinho andHenrissat, 1999, The modular structure of cellulases and othercarbohydrate-active enzymes: an integrated database approach. In“Genetics, Biochemistry and Ecology of Cellulose Degradation”, Ohmiya,Hayashi, Sakka, Kobayashi, Karita and Kimura eds., Uni Publishers Co.,Tokyo, pp. 15-23, and Bourne and Henrissat, 2001, Glycoside hydrolasesand glycosyltransferases: families and functional modules, CurrentOpinion in Structural Biology 11: 593-600.

Examples of enzymes which comprise a CBM suitable for use in the contextof the invention are alpha-amylases, maltogenic alpha-amylases,cellulases, xylanases, mannanases, arabinofuranosidases, acetylesterasesand chitinases. Further CBMs of interest in relation to the presentinvention include CBMs deriving from glucoamylases (EC 3.2.1.3) or fromCGTases (EC 2.4.1.19).

CBMs deriving from fungal, bacterial or plant sources will generally besuitable for use in the context of the invention. Preferred are CBMs offungal origin, more preferably from Aspergillus sp., Bacillus sp.,Klebsiella sp., or Rhizopus sp. In this connection, techniques suitablefor isolating the relevant genes are well known in the art.

Preferred for the invention is CBMs of Carbohydrate-Binding ModuleFamily 20. CBMs of Carbohydrate-Binding Module Family 20 suitable forthe invention may be derived from glucoamylases of Aspergillus awamori(SWISSPROT Q12537), Aspergillus kawachii (SWISSPROT P23176), Aspergillusniger (SWISSPROT P04064), Aspergillus oryzae (SWISSPROT P36914), fromalpha-amylases of Aspergillus kawachii (EMBL:#AB008370), Aspergillusnidulans (NCBI AAF17100.1), from beta-amylases of Bacillus cereus(SWISSPROT P36924), or from CGTases of Bacillus circulans (SWISSPROTP43379). Preferred is a CBM from the alpha-amylase of Aspergilluskawachii (EMBL:#AB008370) as well as CBMs having at least 50%, 60%, 70%,80% or even at least 90% homology to the CBM of the alpha-amylase ofAspergillus kawachii (EMBL:#AB008370), i.e., a CBM having at least 50%,60%, 70%, 80% or even at least 90% homology to the amino acid sequenceof SEQ ID NO:6. Also preferred for the invention are the CBMs ofCarbohydrate-Binding Module Family 20 having the amino acid sequencesshown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11 and disclosed inDanish application no. PA 2003 00949 as SEQ ID NO:1, SEQ ID NO:2 and SEQID NO:3 respectively. Further preferred CBMs include the CBMs of theglucoamylase from Hormoconis sp. such as from Hormoconis resinae (Syn.Creosote fungus or Amorphotheca resinae) such as the CBM ofSWISSPROT:Q03045 (SEQ ID NO:12), from Lentinula sp. such as fromLentinula edodes (shiitake mushroom) such as the CBM of SPTREMBL:Q9P4C5(SEQ ID NO:13), from Neurospora sp. such as from Neurospora crassa suchas the CBM of SWISSPROT:P14804 (SEQ ID NO:14), from Talaromyces sp. suchas from Talaromyces byssochlamydioides such as the CBM of NN005220 (SEQID NO:15), from Geosmithia sp. such as from Geosmithia cylindrospora,such as the CBM of NN48286 (SEQ ID NO:16), from Scorias sp. such as fromScorias spongiosa such as the CBM of NN007096 (SEQ ID NO:17), fromEupenicillium sp. such as from Eupenicillium ludwigii such as the CBM ofNN005968 (SEQ ID NO:18), from Aspergillus sp. such as from Aspergillusjaponicus such as the CBM of NN001136 (SEQ ID NO:19), from Penicilliumsp. such as from Penicillium cf. miczynskii such as the CBM of NN48691(SEQ ID NO:20), from Mz1 Penicillium sp. such as the CBM of NN48690 (SEQID NO:21), from Thysanophora sp. such as the CBM of NN48711 (SEQ IDNO:22), and from Humicola sp. such as from Humicola grisea var.thermoidea such as the CBM of SPTREMBL:Q12623 (SEQ ID NO:23). Mostpreferred CBMs include the CBMs of the glucoamylase from Aspergillus sp.such as from Aspergillus niger, such as SEQ ID NO:24, and Athelia sp.such as from Athelia rolfsii, such as SEQ ID NO:25. Also preferred forthe invention is any CBD having at least 50%, 60%, 70%, 80% or even atleast 90% homology to any of the afore mentioned CBD amino acidsequences.

Further suitable CBMs of Carbohydrate-Binding Module Family 20 may befound at URL: afmb.cnrs-mrs.fr/˜cazy/CAZY/index.html).

Once a nucleotide sequence encoding the substrate-binding(carbohydrate-binding) region has been identified, either as cDNA orchromosomal DNA, it may then be manipulated in a variety of ways to fuseit to a DNA sequence encoding the enzyme of interest. The DNA fragmentencoding the carbohydrate-binding amino acid sequence, and the DNAencoding the enzyme of interest are then ligated with or without alinker. The resulting ligated DNA may then be manipulated in a varietyof ways to achieve expression.

Alpha-Amylolytic Sequence

Alpha-amylases (in particular acid stable alpha-amylases) which areappropriate as the basis for CBM/amylase hybrids of the types employedin the context of the present invention include those of fungal origin.

Preferably the alpha-amylase is a wild type enzyme. More preferably thealpha-amylase is a variant alpha-amylases comprising amino acidmodifications leading to increased activity, increased protein stabilityat low pH, and/or at high pH, increased stability towards calciumdepletion, and/or increased stability at elevated temperature.Chemically or genetically modified mutants of such alpha-amylases areincluded in this connection.

Relevant alpha-amylases include, for example, alpha-amylases obtainablefrom Aspergillus species, in particular from Aspergillus niger, such asan acid stable alpha-amylase (SWISSPROT P56271), described in moredetail in WO 8901969 (example 3) and having the amino acid sequenceshown in SEQ ID NO:8 and/or encode by the DNA sequence shown in SEQ IDNO:7. Also preferred are alpha-amylase sequences having more than 50%,such as 60%, or 70%, 80% or 90% homology to the amino acid sequenceshown in SEQ ID NO:8 and/or encode by the DNA sequence shown in SEQ IDNO:7.

In another preferred embodiment the alpha-amylolytic sequence is derivedfrom the A. oryzae acid alpha-amylase (Fungamyl™). More preferably thealpha-amylolytic sequence has more than 50%, such as 60%, or 70%, 80% or90% homology to the amino acid sequence shown in SEQ ID NO:30 and/or tothe sequence shown as amino acids 21-498 of the amino acid sequenceshown in SEQ ID NO:30.

Even more preferred is an embodiment wherein the hybrid enzyme comprisesan alpha-amylolytic sequence derived from the A. oryzae acidalpha-amylase (Fungamyl™, SEQ ID NO:30), and/or a linker sequencederived from the A. kawachii alpha-amylase or the A. rolfsii AMG, and/ora CBM derived from the A. kawachii alpha-amylase (SEQ ID NO:5) or the A.rolfsii AMG (SEQ ID NO:25). In a particular such embodiment the hybridenzyme has the amino acid sequence shown in SEQ ID NO:36 or in SEQ IDNO:40.

Also preferred is an embodiment wherein the hybrid enzyme comprises analpha-amylolytic sequence derived from the A. niger acid alpha-amylasecatalytic module having the sequence shown in SEQ ID NO:8, and/or alinker sequence derived from the A. kawachii alpha-amylase or the A.rolfsii AMG, and/or the CBM is derived from the A. kawachiialpha-amylase, the A. rolfsii AMG or the A. niger AMG. In a particularlypreferred embodiment the hybrid enzyme comprises the A. niger acidalpha-amylase catalytic module having the sequence shown in SEQ ID NO:8and the A. kawachii alpha-amylase linker and CBM (SEQ ID NO:6).

Preferably the hybrid enzyme comprises a CBD sequence having at least50%, 60%, 70%, 80% or even at least 90% homology to any of the aminoacid sequences shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25. Even more preferredthe hybrid enzyme comprises a CBD sequence having an amino acid sequenceshown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24 or SEQ ID NO:25. In yet another preferred embodimentthe CBM sequence has an amino acid sequence which differs from the aminoacid sequence amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25in no more than 10 amino acid positions, no more than 9 positions, nomore than 8 positions, no more than 7 positions, no more than 6positions, no more than 5 positions, no more than 4 positions, no morethan 3 positions, no more than 2 positions, or even no more than 1position. In a most preferred embodiment the hybrid enzyme comprises aCBM derived from an AMG from A. rolfsii, such as the AMG from A. rolfsiiAHU 9627 described in U.S. Pat. No. 4,727,026.

In a particular embodiment the hybrid enzyme has the amino acid sequenceshown in SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:38 or the hybrid enzymehas an amino acid sequence having at least 50%, 60%, 70%, 80% or even atleast 90% homology to any of the afore mentioned amino acid sequences.

In yet another preferred embodiment the hybrid enzyme has an amino acidsequence which differs from the amino acid sequence amino acid sequenceshown in SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38 or SEQID NO:40 in no more than 10 positions, no more than 9 positions, no morethan 8 positions, no more than 7 positions, no more than 6 positions, nomore than 5 positions, no more than 4 positions, no more than 3positions, no more than 2 positions, or even no more than 1 position.

In a preferred embodiment the hybrid enzyme is a variant comprises thecatalytic domain shown as amino acids 21-498 of SEQ ID NO:40 (and/oramino acids 21-498 of SEQ ID NO:30) with one or more substitutions, morepreferably a substitution in one or more positions selected from thelist consisting of: 81, 158, 161, 163, 164, 175, 176, 177, 253, 264,266, 466, 468, 470, and most preferably one or more substitutionsselected from the list consisting of: □81R, K158D, K158V, S161 D, S161N, Q163S, Q163A, D164S, Y175W, E176D, D177N, D253N,N264K, N264E, M266L,G466D, D468S, and N470D. In an even more preferred embodiment the hybridenzyme is one of the variants listed in Table 6. Also preferred arevariants comprising the catalytic domain shown as amino acids 21-498 ofSEQ ID NO:40 (and/or amino acids 21-498 of SEQ ID NO:30) with one ormore substations or combination of substitutions selected from the listcomprising: Y175W+E176D, 276W+P277Q, K380T+L381W, G62N, G62F, Y95D,D106K+S109D, A140P, K158D+D164S, F171L+E182Q, K200E+V202A, K200E+V202A,K200E+V202A, T227S+K233P, L252D+D255N, N264K+M266L, K283E, N359K+D360V,N390K+Y391L, G466D+N470D, Y484L+S498S, Y484L+S498G. and Y484L+S498R.

In a preferred embodiment the hybrid enzyme comprises the catalyticmodule according shown as amino acids 21-498 of SEQ ID NO:40 (and/oramino acids 21-498 of SEQ ID NO:30) with one or more substitutionsselected from the list consisting of: K158V, S161 N, Q163A, D164S,N264K, M266L, G466D, D468S and N470D.

In another preferred embodiment the hybrid enzyme comprises thecatalytic module shown as amino acids 21-498 of SEQ ID NO:40 (and/oramino acids 21-498 of SEQ ID NO:30) with one or more substitutionsselected from the list consisting of: □81R, K158V, S161 N, Q163A, D164S,Y175W, E176D, D177N, N264K, M266L, G466D, D468S and N470D.

Expression Vectors

The present invention also relates to recombinant expression vectorswhich may comprise a DNA sequence encoding the hybrid enzyme or agenetically modified wild type enzyme, a promoter, a signal peptidesequence, and transcriptional and translational stop signals. Thevarious DNA and control sequences described above may be joined togetherto produce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe DNA sequence encoding the polypeptide at such sites. Alternatively,the DNA sequence of the present invention may be expressed by insertingthe DNA sequence or a DNA construct comprising the sequence into anappropriate vector for expression. In creating the expression vector,the coding sequence is located in the vector so that the coding sequenceis operably linked with the appropriate control sequences forexpression, and possibly secretion.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the DNA sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids. The vector may be anautonomously replicating vector, i.e., a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a plasmid, an extrachromosomal element, aminichromosome, a cosmid or an artificial chromosome. The vector maycontain any means for assuring self-replication. Alternatively, thevector may be one which, when introduced into the host cell, isintegrated into the genome and replicated together with thechromosome(s) into which it has been integrated. The vector system maybe a single vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon.

Markers

The vectors of the present invention preferably contain one or moreselectable markers, which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like.

Examples of selectable markers for use in a filamentous fungus host cellmay be selected from the group including, but not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), and glufosinate resistance markers, aswell as equivalents from other species. Preferred for use in anAspergillus cell are the amdS and pyrG markers of Aspergillus nidulansor Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus.Furthermore, selection may be accomplished by co-transformation, e.g.,as described in WO 91/17243, where the selectable marker is on aseparate vector.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell genomeor autonomous replication of the vector in the cell independent of thegenome of the cell.

The vectors of the present invention may be integrated into the hostcell genome when introduced into a host cell. For integration, thevector may rely on the DNA sequence encoding the polypeptide of interestor any other element of the vector for stable integration of the vectorinto the genome by homologous or none homologous recombination.Alternatively, the vector may contain additional DNA sequences fordirecting integration by homologous recombination into the genome of thehost cell. The additional DNA sequences enable the vector to beintegrated into the host cell genome at a precise location(s) in thechromosome(s). To increase the likelihood of integration at a preciselocation, the integrational elements should preferably contain asufficient number of DNAs, such as 100 to 1,500 base pairs, preferably400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs,which are highly homologous with the corresponding target sequence toenhance the probability of homologous recombination. The integrationalelements may be any sequence that is homologous with the target sequencein the genome of the host cell. Furthermore, the integrational elementsmay be non-encoding or encoding DNA sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination. These DNA sequences may be any sequencethat is homologous with a target sequence in the genome of the hostcell, and, furthermore, may be non-encoding or encoding sequences.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question.

The episomal replicating the AMA1 plasmid vector disclosed in WO00/24883 may be used.

More than one copy of a DNA sequence encoding a polypeptide of interestmay be inserted into the host cell to amplify expression of the DNAsequence. Stable amplification of the DNA sequence can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome using methods well known in the art and selecting fortransformants.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al, 1989,Molecular Cloning, A Laboratory Manual, 2^(nd) edition, Cold SpringHarbor, N.Y.).

Host Cells

The host cell of the invention, either comprising a DNA construct or anexpression vector comprising the DNA sequence encoding the hybrid enzymeor a genetically modified wild type enzyme, is advantageously used as ahost cell in the recombinant production of the hybrid enzyme or agenetically modified wild type enzyme. The cell may be transformed withan expression vector. Alternatively, the cell may be transformed withthe DNA construct of the invention encoding the hybrid enzyme or agenetically modified wild type enzyme, conveniently by integrating theDNA construct (in one or more copies) in the host chromosome.Integration of the DNA construct into the host chromosome may beperformed according to conventional methods, e.g., by homologous orheterologous recombination.

The host cell may be any appropriate prokaryotic or eukaryotic cell,e.g., a bacterial cell, a filamentous fungus cell, a plant cell or amammalian cell.

In a preferred embodiment, the host cell is a filamentous fungusrepresented by the following groups of Ascomycota, include, e.g.,Neurospora, Eupenicillium (=Penicillium), Emericella (=Aspergillus),Eurotium (=Aspergillus).

In a more preferred embodiment, the filamentous fungus include allfilamentous forms of the subdivision Eumycota and Oomycota (as definedby Hawksworth et al. In, Ainsworth and Bisby's Dictionary of The Fungi,8^(th) edition, 1995, CAB International, University Press, Cambridge,UK. The filamentous fungi are characterized by a vegetative myceliumcomposed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligately aerobic.

In an even more preferred embodiment, the filamentous fungus host cellis a cell of a species of, but not limited to a cell selected from thegroup consisting of a strain belonging to a species of Aspergillus,preferably Aspergillus oryzae, Aspergillus niger, Aspergillus awamori,Aspergillus kawachii, or a strain of Bacillus, or a strain of Fusarium,such as a strain of Fusarium oxysporium, Fusarium graminearum (in theperfect state named Gribberella zeae, previously Sphaeria zeae, synonymwith Gibberella roseum and Gibberella roseum f. sp. cerealis), orFusarium sulphureum (in the prefect state named Gibberella puricaris,synonym with Fusarium trichothecioides, Fusarium bactridioides, Fusariumsambucium, Fusarium roseum, and Fusarium roseum var. graminearum),Fusarium cerealis (synonym with Fusarium crookwellense), or Fusariumvenenatum.

In a most preferred embodiment, the filamentous fungus host cell is acell of a strain belonging to a species of Aspergillus, preferablyAspergillus oryzae or Aspergillus niger.

The host cell may be a wild type filamentous fungus host cell or avariant, a mutant or a genetically modified filamentous fungus hostcell. In a preferred embodiment of the invention the host cell is aprotease deficient or protease minus strain. Also specificallycontemplated is Aspergillus strains, such as Aspergillus niger strains,genetically modified to disrupt or reduce expression of glucoamylase,acid-stable alpha-amylase, alpha-1,6 transglucosidase, and proteaseactivities.

Transformation of Filamentous Fungus Host Cells

Filamentous fungus host cells may be transformed by a process involvingprotoplast formation, transformation of the protoplasts, andregeneration of the cell wall in a manner known in the art. Suitableprocedures for transformation of Aspergillus host cells are described inEP 238 023, EP 184 438, and Yelton et al., 1984, Proceedings of theNational Academy of Sciences USA 81:1470-1474. A suitable method oftransforming Fusarium species is described by Malardier et al., 1989,Gene 78:147-156 or U.S. Pat. No. 6,060,305.

Isolating and Cloning a DNA Sequence Encoding a Parent Alpha-Amylase

The techniques used to isolate or clone a DNA sequence encoding apolypeptide of interest are known in the art and include isolation fromgenomic DNA, preparation from cDNA, or a combination thereof. Thecloning of the DNA sequences of the present invention from such genomicDNA can be effected, e.g., by using the well known polymerase chainreaction (PCR) or antibody screening of expression libraries to detectcloned DNA fragments with shared structural features. See, e.g., Inniset al., 1990, PCR: A Guide to Methods and Application, Academic Press,New York. Other DNA amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and DNAsequence-based amplification (NASBA) may be used.

The DNA sequence encoding a parent alpha-amylase may be isolated fromany cell or microorganism producing the alpha-amylase in question, usingvarious methods well known in the art. First, a genomic DNA and/or cDNAlibrary should be constructed using chromosomal DNA or messenger RNAfrom the organism that produces the alpha-amylase to be studied. Then,if the amino acid sequence of the alpha-amylase is known, labeledoligonucleotide probes may be synthesized and used to identifyalpha-amylase-encoding clones from a genomic library prepared from theorganism in question. Alternatively, a labelled oligonucleotide probecontaining sequences homologous to another known alpha-amylase genecould be used as a probe to identify alpha-amylase-encoding clones,using hybridization and washing conditions of very low to very highstringency.

Yet another method for identifying alpha-amylase-encoding clones wouldinvolve inserting fragments of genomic DNA into an expression vector,such as a plasmid, transforming alpha-amylase-negative bacteria with theresulting genomic DNA library, and then plating the transformed bacteriaonto agar containing a substrate for alpha-amylase (i.e., maltose),thereby allowing clones expressing the alpha-amylase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g., thephosphoroamidite method described in Beaucage and Caruthers, 1981,Tetrahedron Letters 22: 1859-1869, or the method described by Matthes etal., 1984, EMBO J. 3: 801-805. In the phosphoroamidite method,oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer,purified, annealed, ligated and cloned in appropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate, the fragments corresponding to various parts of the entireDNA sequence), in accordance with standard techniques. The DNA sequencemay also be prepared by polymerase chain reaction (PCR) using specificprimers, for instance as described in U.S. Pat. No. 4,683,202 or Saikiet al., 1988, Science 239: 487-491.

Isolated DNA Sequence

The present invention relates, inter alia, to an isolated DNA sequencecomprising a DNA sequence encoding a hybrid enzyme or a geneticallymodified wild type enzyme comprising an amino acid sequence of acatalytic module having alpha-amylase activity and an amino acidsequence of a carbohydrate-binding module, wherein the catalytic moduleis of fungal origin.

The term “isolated DNA sequence” as used herein refers to a DNAsequence, which is essentially free of other DNA sequences, e.g., atleast about 20% pure, preferably at least about 40% pure, morepreferably at least about 60% pure, even more preferably at least about80% pure, and most preferably at least about 90% pure as determined byagarose electrophoresis.

For example, an isolated DNA sequence can be obtained by standardcloning procedures used in genetic engineering to relocate the DNAsequence from its natural location to a different site where it will bereproduced. The cloning procedures may involve excision and isolation ofa desired DNA fragment comprising the DNA sequence encoding thepolypeptide of interest, insertion of the fragment into a vectormolecule, and incorporation of the recombinant vector into a host cellwhere multiple copies or clones of the DNA sequence will be replicated.An isolated DNA sequence may be manipulated in a variety of ways toprovide for expression of the polypeptide of interest. Manipulation ofthe DNA sequence prior to its insertion into a vector may be desirableor necessary depending on the expression vector. The techniques formodifying DNA sequences utilizing recombinant DNA methods are well knownin the art.

DNA Construct

The present invention relates, inter alia, to a DNA construct comprisinga DNA sequence encoding a hybrid enzyme or a genetically modified wildtype enzyme comprising an amino acid sequence of a catalytic modulehaving alpha-amylase activity and an amino acid sequence of acarbohydrate-binding module, wherein the catalytic module is of fungalorigin. “DNA construct” is defined herein as a DNA molecule, eithersingle- or double-stranded, which is isolated from a naturally occurringgene or which has been modified to contain segments of DNA, which arecombined and juxtaposed in a manner, which would not otherwise exist innature. The term DNA construct is synonymous with the term expressioncassette when the DNA construct contains all the control sequencesrequired for expression of a coding sequence of the present invention.

Site-Directed Mutagenesis

Once a parent alpha-amylase-encoding DNA sequence has been isolated, anddesirable sites for mutation identified, mutations may be introducedusing synthetic oligonucleotides. These oligonucleotides containnucleotide sequences flanking the desired mutation sites. In a specificmethod, a single-stranded gap of DNA, the alpha-amylase-encodingsequence, is created in a vector carrying the alpha-amylase gene. Thenthe synthetic nucleotide, bearing the desired mutation, is annealed to ahomologous portion of the single-stranded DNA. The remaining gap is thenfilled in with DNA polymerase I (Klenow fragment) and the construct isligated using T4 ligase. A specific example of this method is describedin Morinaga et al., 1984, Biotechnology 2: 646-639. U.S. Pat. No.4,760,025 discloses the introduction of oligonucleotides encodingmultiple mutations by performing minor alterations of the cassette.However, an even greater variety of mutations can be introduced at anyone time by the Morinaga method, because a multitude ofoligonucleotides, of various lengths, can be introduced.

Another method for introducing mutations into alpha-amylase-encoding DNAsequences is described in Nelson and Long, 1989, Analytical Biochemistry180: 147-151. It involves the 3-step generation of a PCR fragmentcontaining the desired mutation introduced by using a chemicallysynthesized DNA strand as one of the primers in the PCR reactions. Fromthe PCR-generated fragment, a DNA fragment carrying the mutation may beisolated by cleavage with restriction endonucleases and reinserted intoan expression plasmid.

Localized Random Mutagenesis

The random mutagenesis may be advantageously localized to a part of theparent alpha-amylase in question. This may, e.g., be advantageous whencertain regions of the enzyme have been identified to be of particularimportance for a given property of the enzyme, and when modified areexpected to result in a variant having improved properties. Such regionsmay normally be identified when the tertiary structure of the parentenzyme has been elucidated and related to the function of the enzyme.

The localized or region-specific, random mutagenesis is convenientlyperformed by use of PCR generated mutagenesis techniques as describedabove or any other suitable technique known in the art. Alternatively,the DNA sequence encoding the part of the DNA sequence to be modifiedmay be isolated, e.g., by insertion into a suitable vector, and saidpart may be subsequently subjected to mutagenesis by use of any of themutagenesis methods discussed above.

Variants of Hybrid or Wild Type Enzymes

The performance in a starch degradation process of a wild type or hybridenzyme comprising a carbohydrate-binding module (“CBM”) and analpha-amylase catalytic module may be improved through proteinengineering, such as by site directed mutagenesis, by localized randommutagenesis, by synthetically preparing a new variant of the parent wildtype enzyme or parent hybrid enzyme, or by any other suitable proteinengineering techniques.

Parent alpha-amylase contemplated for the invention include wild-typefungal alpha-amylase having a CBM, in particular fungal alpha-amylaseobtainable from an Aspergillus strain, such as an Aspergillus kawachiiacid alpha-amylase and variants or mutants thereof, homologousalpha-amylases, and further wild type or artificial alpha-amylases beingstructurally and/or functionally similar to the acid alpha-amylase fromAspergillus kawachii shown in SEQ ID NO:41.

While the acid alpha-amylase from Aspergillus kawachii is an example ofa wild type alpha-amylase comprising a carbohydrate-binding module(“CBM”), the Aspergillus kawachii acid alpha-amylase catalytic modulehas a limited specific activity, e.g., compared to the catalyticactivity of the acid alpha-amylase from A. niger. Furthermore, the CBMof the Aspergillus kawachii alpha-amylase is easily cleaved off from thecatalytic module during a conventional fermentation process therebyreducing the suitability of the enzyme for industrial application. Thusthe Aspergillus kawachii alpha-amylase has several serious drawbacks asan industrial enzyme for raw starch hydrolysis. Protein engineeringtechniques can be applied to yield more suitable variants of a parentacid alpha-amylase wherein the catalytic activity and/or the enzymestability has been improved.

Preferred variants are variants of the fungal alpha-amylase shown as SEQID NO:41 or variants of an alpha-amylase having at least 60% homology,at least 70% homology, at least 80% homology, or even at least 90%homology to SEQ ID NO:41, which variant comprising an alteration at oneor more of the positions: 13, 15, 18, 31, 32, 33, 34, 35, 36, 61, 63,64, 68, 69, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 89, 117,118, 119, 120, 121, 122, 123, 124, 125, 152, 153, 154, 155, 156, 157,158, 161, 162, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,204, 205, 206, 207, 208, 209, 210, 211, 216, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 242, 245, 250, 252, 253, 255, 256, 257,259, 260, 275, 292, 295, 296, 297, 298, 299, 304, 328, 339, 344, 348,378, 383, 386, 387, 405, 448 and 480 and more preferred at one or moreof the positions 31, 33, 36, 74, 75, 77, 84, 120, 153, 154, 155, 156,157, 158, 162, 166, 169, 170, 199, 232, 233, 235, 238, 239, 245, 256,257, 331, 336, 339, 340, 342, 348, 378, 383, 386, 387, 405, 448 and 480wherein (a) the alteration(s) are independently (i) an insertion of anamino acid downstream of the amino acid which occupies the position,(ii) a deletion of the amino acid which occupies the position, or iii) asubstitution of the amino acid which occupies the position with adifferent amino acid, (b) the variant has increased acid alpha-amylaseactivity and or improved enzyme stability relative to the parent fungalalpha-amylase and (c) each position corresponds to a position of theamino acid sequence of the TAKA amylase shown in SEQ ID NO:43 and/or theA. kawachii alpha-amylase shown as SEQ ID NO:41.

More preferred variants are variants of the A. kawachii alpha-amylaseshown as SEQ ID NO:41 or variants of an alpha-amylase having at least60% homology, at least 70% homology, at least 80% homology, or even atleast 90% homology to SEQ ID NO:41 and comprising alterations in one ormore of the following positions: 13, 15, 18, 31, 32, 33, 34, 35, 36, 61,63, 64, 68, 69, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 89, 117,118, 119, 120, 121, 122, 123, 124, 125, 152, 153, 154, 155, 156, 157,158, 161, 162, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,204, 205, 206, 207, 208, 209, 210, 211, 216, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 242, 245, 250, 252, 253, 255, 256, 257,259, 260, 275, 292, 295, 296, 297, 298, 299, 304, 328, 339, 344, 348,378, 383, 386, 387, 405, 448 and 480 wherein (a) the alteration(s) areindependently (i) an insertion of an amino acid downstream of the aminoacid which occupies the position, (ii) a deletion of the amino acidwhich occupies the position, or iii) a substitution of the amino acidwhich occupies the position with a different amino acid, (b) the varianthas increased acid alpha-amylase activity and or improved enzymestability relative to the parent fungal alpha-amylase and (c) eachposition corresponds to a position of the amino acid sequence of theTAKA amylase shown in SEQ ID NO:43 and/or the A. kawachii alpha-amylaseshown as SEQ ID NO:41. Even more preferred variants of the A. kawachiialpha-amylase shown as SEQ ID NO:41 or variants of an alpha-amylasehaving at least 60% homology, at least 70% homology, at least 80%homology, or even at least 90% homology to SEQ ID NO:41 and comprisingalterations in one or more of the following positions: 31, 33, 36, 74,75, 77, 84, 120, 153, 154, 155, 156, 157, 158, 162, 166, 169, 170, 199,232, 233, 235, 238, 239, 245, 256, 257, 331, 336, 339, 340, 342, 348,378, 383, 386, 387, 405, 448 and 480.

Yet more preferred are variants of the A. kawachii alpha-amylase shownas SEQ ID NO: 41 or variants of an alpha-amylase having at least 60%homology, at least 70% homology, at least 80% homology, or even at least90% homology to SEQ ID NO: 41 comprising one or more of the followingamino acid substitution: G33A, 136K, S74A, D75Y, E77D, P120A, I153D,D154N, W155Y, D156E, N157D, L158Q, Q162E, E166L, T169N, I170T, E199K,E199L, D232L, N233D, N235D, L238Y, D239T, W256Y, Q257P, E331Q, S336A,D339K, D339N, V340D, and Y342A, and most preferred variants comprisesone or more of the following amino acid substitution S74A, E166L, E199L,D339K, and D156E, such as the variant comprising the multiple amino acidsubstitution: S74A/E166L/E199L, wherein the variant has improve activityand/or enzyme stability relative to the parent alpha-amylase shown asSEQ ID NO:41.

The variants may also be a variants of the A. kawachii alpha-amylaseshown as SEQ ID NO:41 or variants of an alpha-amylase having at least60% homology, at least 70% homology, at least 80% homology, or even atleast 90% homology to SEQ ID NO:41 and comprising alterations in one ormore of the following positions: 31, 74, 89, 209, 245, 348, 378, 383,386, 387, 405, 448, and 480, wherein (a) the alteration(s) areindependently (i) an insertion of an amino acid downstream of the aminoacid which occupies the position, (ii) a deletion of the amino acidwhich occupies the position, or iii) a substitution of the amino acidwhich occupies the position with a different amino acid, (b) the varianthas increased acid alpha-amylase activity and or improved enzymestability relative to the parent fungal alpha-amylase.

Preferably the variants is a variants of the A. kawachii alpha-amylaseshown as SEQ ID NO:41 or variants of an alpha-amylase having at least60% homology, at least 70% homology, at least 80% homology, or even atleast 90% homology to SEQ ID NO:41 and comprising one or more of thefollowing substitutions: N31D, S74A, Y89D, E209L, Y245V, D348K, D378A,K383A, P386A, I387F, I405V, N448S, and N480R, wherein the variant hasincreased acid alpha-amylase activity and or improved enzyme stabilityrelative to the parent fungal alpha-amylase shown as SEQ ID NO:41.

Most preferred is a variants of the A. kawachii alpha-amylase shown asSEQ ID NO:41 and comprising the following substitutions: N31D, S74A,Y89D, E209L, Y245V, D348K, D378A, K383A, P386A, I387F, I405V, N448S, andN480R.

The variants may be produced using conventional protein engineeringtechniques.

Expression of the Enzymes in Plants

A DNA sequence encoding an enzyme of interest, such as a hybrid enzymeor a variant of a wild type enzyme or a hybrid of the present invention,may be transformed and expressed in transgenic plants as describedbelow.

The transgenic plant can be dicotyledonous or monocotyledonous, forshort a dicot or a monocot. Examples of monocot plants are grasses, suchas meadow grass (blue grass, Poa), forage grass such as Festuca, Lolium,temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye,barley, rice, sorghum and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, oil seed rape and the closelyrelated model organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems. Inthe present context, also specific plant cell compartments, such aschloroplast, apoplast, mitochondria, vacuole, peroxisomes and cytoplasmare considered to be a plant part. Furthermore, any plant cell, whateverthe tissue origin, is considered to be a plant part. Likewise, plantparts such as specific tissues and cells isolated to facilitate theutilisation of the invention are also considered plant parts, e.g.,embryos, endosperms, aleurone and seeds coats.

Also included within the scope of the invention are the progeny of suchplants, plant parts and plant cells.

The transgenic plant or plant cell expressing the enzyme of interest maybe constructed in accordance with methods known in the art. In short theplant or plant cell is constructed by incorporating one or moreexpression constructs encoding the enzyme of interest into the planthost genome and propagating the resulting modified plant or plant cellinto a transgenic plant or plant cell.

Conveniently, the expression construct is a DNA construct whichcomprises a gene encoding the enzyme of interest in operable associationwith appropriate regulatory sequences required for expression of thegene in the plant or plant part of choice. Furthermore, the expressionconstruct may comprise a selectable marker useful for identifying hostcells into which the expression construct has been integrated and DNAsequences necessary for introduction of the construct into the plant inquestion (the latter depends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences is determined,e.g., on the basis of when, where and how the enzyme is desired to beexpressed. For instance, the expression of the gene encoding the enzymeof the invention may be constitutive or inducible, or may bedevelopmental, stage or tissue specific, and the gene product may betargeted to a specific cell compartment, tissue or plant part such asseeds or leaves. Regulatory sequences are, e.g., described by Tague etal., 1988, Plant Phys. 86: 506.

For constitutive expression the 35S-CaMV, the maize ubiquitin 1 and therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294, Christensen, Sharrock and Quail, 1992, Maize polyubiquitingenes: structure, thermal perturbation of expression and transcriptsplicing, and promoter activity following transfer to protoplasts byelectroporation. Plant Mol. Biol. 18: 675-689; Zhang, McElroy and Wu,1991, Analysis of rice Act1 5′ region activity in transgenic riceplants. Plant Cell 3: 1155-1165). Organ-specific promoters may, e.g., bea promoter from storage sink tissues such as seeds, potato tubers, andfruits (Edwards and Coruzzi, 1990, Annu. Rev. Genet. 24: 275-303), orfrom metabolic sink tissues such as meristems (Ito et al., 1994, PlantMol. Biol. 24: 863-878), a seed specific promoter such as the glutelin,prolamin, globulin or albumin promoter from rice (Wu et al., 1998, Plantand Cell Physiology 39(8): 885-889), a Vicia faba promoter from thelegumin B4 and the unknown seed protein gene from Vicia faba describedby Conrad et al., 1998, Journal of Plant Physiology 152(6): 708-711, apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39(9): 935-941, the storage protein napA promoter fromBrassica napus, or any other seed specific promoter known in the art,e.g., as described in WO 91/14772. Furthermore, the promoter may be aleaf specific promoter such as the rbcs promoter from rice or tomato(Kyozuka et al., 1993, Plant Physiology 102(3): 991-1000, the chlorellavirus adenine methyltransferase gene promoter (Mitra and Higgins, 1994,Plant Molecular Biology 26(1): 85-93, or the aldP gene promoter fromrice (Kagaya et al., 1995, Molecular and General Genetics 248(6):668-674, or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Molecular Biology 22(4): 573-588. Likewise, thepromoter may inducible by abiotic treatments such as temperature,drought or alterations in salinity or induced by exogenously appliedsubstances that activate the promoter e.g., ethanol, oestrogens, planthormones like ethylene, abscisic acid and gibberellic acid and heavymetals.

A promoter enhancer element may be used to achieve higher expression ofthe enzyme in the plant. For instance, the promoter enhancer element maybe an intron which is placed between the promoter and the nucleotidesequence encoding the enzyme. For instance, Xu et al. op cit disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The DNA construct is incorporated into the plant genome according toconventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,micro injection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., Science 244: 1293; Potrykus, 1990,Bio/Techn. 8: 535; and Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens mediated gene transfer is themethod of choice for generating transgenic dicots (for review Hooykas &Schilperoort, 1992, Plant Mol. Biol. 19: 15-38), and can also be usedfor transforming monocots, although other transformation methods oftenare used for these plants. Presently, the method of choice forgenerating transgenic monocots supplementing the Agrobacterium approachis particle bombardment (microscopic gold or tungsten particles coatedwith the transforming DNA) of embryonic calli or developing embryos(Christou, 1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin.Biotechnol. 5: 158-162; Vasil et al., 1992, Bio/Technology 10: 667-674).An alternative method for transformation of monocots is based onprotoplast transformation as described by Omirulleh et al., 1993, PlantMolecular Biology 21(3): 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using,e.g., co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

Starch Processing

The hybrid enzyme of the first aspect of the invention or the variant ofthe second aspect of the invention may in an eighth aspect be used in aprocess for liquefying starch, wherein a gelatinized or granular starchsubstrate is treated in aqueous medium with the hybrid enzyme.Preferably the process comprising hydrolysis of a slurry of gelatinizedor granular starch, in particular hydrolysis of granular starch into asoluble starch hydrolysate at a temperature below the initialgelatinization temperature of said granular starch.

In a preferred embodiment the starch slurry in addition to beingcontacted with the hybrid enzyme of the first aspect or the variant ofthe second aspect of the invention is contacted with an enzyme selectedfrom the list consisting of; a fungal alpha-amylase (EC 3.2.1.1), abeta-amylase (E.C. 3.2.1.2), and a glucoamylase (E.C.3.2.1.3). In anembodiment further a Termamyl-like alpha-amylase or a debranchingenzyme, such as an isoamylase (E.C. 3.2.1.68) or a pullulanases (E.C.3.2.1.41) is added. In the context of the present invention aTermamyl-like alpha-amylase is an alpha-amylase as defined in WO99/19467 on page 3, line 18 to page 6, line 27.

The starch slurry to be subjected to the process of the seventh aspectof the invention may have 20-55% dry solids granular starch, preferably25-40% dry solids granular starch, more preferably 30-35% dry solidsgranular starch.

After being subjected to the process of the seventh aspect of theinvention at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, orpreferably at least 99% of the dry solids of the granular starch isconverted into a soluble starch hydrolysate.

According to the invention the process of the seventh aspect isconducted at a temperature below the initial gelatinization temperature.Preferably the temperature at which the processes are conducted is atleast 30° C., at least 31° C., at least 32° C., at least 33° C., atleast 34° C., at least 35° C., at least 36° C., at least 37° C., atleast 38° C., at least 39° C., at least 40° C., at least 41° C., atleast 42° C., at least 43° C., at least 44° C., at least 45° C., atleast 46° C., at least 47° C., at least 48° C., at least 49° C., atleast 50° C., at least 51° C., at least 52° C., at least 53° C., atleast 54° C., at least 55° C., at least 56° C., at least 57° C., atleast 58° C., at least 59° C., or preferably at least 60° C.

The pH at which the process of the seventh aspect of the invention isconducted may in be in the range of 3.0 to 7.0, preferably from 3.5 to6.0, or more preferably from 4.0-5.0.

The granular starch to be processed in the process of the invention mayin particular be obtained from tubers, roots, stems, legumes, cereals orwhole grain. More specifically the granular starch may be obtained fromcorns, cobs, wheat, barley, rye, milo, sago, cassaya, tapioca, sorghum,rice, peas, bean, banana or potatoes. Specially contemplated are bothwaxy and non-waxy types of corn and barley. The granular starch to beprocessed may be a highly refined starch quality, preferably at least90%, at least 95%, at least 97% or at least 99.5% pure or it may be amore crude starch containing material comprising milled whole grainincluding non-starch fractions such as germ residues and fibres. The rawmaterial, such as whole grain, is milled in order to open up thestructure and allowing for further processing. Two milling processes arepreferred according to the invention: wet and dry milling. In drymilling the whole kernel is milled and used. Wet milling gives a goodseparation of germ and meal (starch granules and protein) and is with afew exceptions applied at locations where the starch hydrolysate is usedin production of syrups. Both dry and wet milling is well known in theart of starch processing and are equally contemplated for the process ofthe invention. Also corn grits, and preferably milled corn grits may beapplied.

In an embodiment of the process of the seventh aspect of the inventionthe hybrid enzyme is used in a process for production of fuel or potableethanol comprising contacting the treated starch with a yeast.Preferably the process comprises fermentation with a yeast carried outsimultaneously or separately/sequential to the hydrolysis of thegranular starch slurry. When the fermentation is performed simultaneousto the hydrolysis the temperature is preferably between 30° C. and 35°C., and more preferably between 31° C. and 34° C.

In another embodiment the granular starch slurry is being contacted witha polypeptide comprising a CBM, but no catalytic module, i.e.,application of loose CBMs. The loose CBMs may be starch binding modules,cellulose-binding modules, chitin-binding modules, xylan-bindingmodules, mannan-binding modules, and other binding modules. PreferredCBMs in the present context are microbial CBMs, particularly bacterialor fungal CBMs. Particularly preferred are the starch binding modulesdisclosed in Danish application no. PA 2003 01568 as the polypeptidesequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 or the starchbinding modules shown in the present disclosure as SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15; SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18; SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23. Most preferred CBMs include the CBMs disclosed in the presentdisclosure as SEQ ID NO:24 and as SEQ ID NO:25. Also preferred for theinvention is the application of any CBM having at least 50%, 60%, 70%,80% or even at least 90% homology to any of the afore mentioned CBMamino acid sequences. The loose CBMs may be applied to the granularstarch slurry in effective amounts.

The glucose may also be fermented in to other fermentation products,such as citric acid, itaconic acid, lactic acid, gluconic acid; ketones;amino acids, such as glutamic acid (sodium monoglutaminate), but alsomore complex compounds such as antibiotics, such as penicillin,tetracyclin; enzymes; vitamins, such as riboflavin, B12, beta-carotene;hormones, which are difficult to produce synthetically.

Dough-Based Products

The hybrid enzyme may be used for the preparation of a dough-basededible product, particularly a hybrid enzyme comprising an amylolyticmodule derived from Aspergillus oryzae such as the amino acid sequenceshown in SEQ ID NO: 30. The hybrid enzyme used for the preparation of adough-based edible product is in particular a hybrid enzyme comprisingthe sequence shown as SEQ ID NO: 30 and/or as amino acids 21-498 of SEQID NO: 30 or any variant thereof such as a variant comprising one ormore substitutions selected from the list consisting of: □81R, K158D,K158V, S161 D, S161 N, Q163S, Q163A, D164S, Y175W, E176D, D177N, N264K,N264E, M266L, G466D, D468S, and N470D. The hybrid enzyme used for thepreparation of a dough-based edible product is in particular a hybridenzyme comprising an amino acid sequence which has at least 50%homology, preferably at least 60%, 70%, 80%, 85% or at least 90%, e.g.,at least 95%, 97%, 98%, or at least 99%, such as 100% homology to thesequences set forth in SEQ ID NO: 30.

The dough generally comprises flour (particularly wheat flour) andwater. The dough is leavened, e.g., by adding chemical leavening agentsor yeast, usually Saccharomyces cerevisiae (baker's yeast).

The dough-based product is made by leavening and heating the dough,e.g., by baking or steaming. Examples are steamed or baked bread (inparticular white, whole-meal or rye bread), typically in the form ofloaves or rolls,

The dough may comprise one or more additional enzymes, e.g., a secondamylase (e.g., a maltogenic alpha-amylase), a cyclodextringlucanotransferase, a protease or peptidase, in particular anexopeptidase, a transglutaminase, a lipase, a phospholipase, acellulase, a hemicellulase (e.g., a pentopsanase or xylanase), aglycosyltransferase, a branching enzyme or an oxidase such as glucoseoxidase or an oxidase with higher activity on maltose than on glucose.

The hybrid enzyme may be used at a lower dosage than the catalyticmodule with alpha-amylase activity used alone (compared on a weightbasis).

Materials and Methods Purchased Material

Enzymes for DNA manipulations (e.g., restriction endonucleases, ligasesetc.) are obtainable from New England Biolabs, Inc. and were usedaccording to the manufacturer's instructions. Amplified plasmids wererecovered with Qiagen® Plasmid Kit (Qiagen). Polymerase Chain Reaction(PCR) was carried out with Expand™ PCR system (Roche). QIAquick™ GelExtraction Kit (Qiagen) was used for purification of PCR fragments andDNA fragments excised from agarose gels.

Strains and Plasmids

Aspergillus kawachii strain IF04308 was used as donor of CBM and linkersequences. Aspergillus niger strain DSM 2761 donated the amylolyticsequence (WO89/01969). Aspergillus niger strain MBin120 described inU.S. provisional patent application No. 60/459,902 (10345.000-US) wasused as host strain. The Aspergillus expression plasmid pMT2188described in the patent WO200295014 was used as vector. The pyrFdefective Escherichia coli strain DB6507 (ATCC 35673) was used forpropagation of pHUda381 and pHUda387

Plasmid pMT2188 consists of an expression cassette based on theAspergillus niger neutral amylase II promoter fused to the Aspergillusnidulans triose phosphate isomerase non translated leader sequence(Pna2/tpi) and the Aspergillus niger amyloglycosidase terminator (Tamg).Also present on the plasmid is the Aspergillus selective marker amdSfrom Aspergillus nidulans enabling growth on acetamide as sole nitrogensource and the URA3 marker from Saccharomyces cerevisiae enabling growthof the pyrF defective Escherichia coli strain DB6507 (ATCC 35673), whichpropagates pHUda381 and pHUda387. Transformation into E. coli DB6507using the S. cerevisiae URA 3 gene as selective marker was done in thefollowing way:

E. coli DB6507 was made competent by the method of Mandel and Higa(Mandel and Higa, 1970, J. Mol. Biol. 45: 154). Transformants wereselected on solid M9 medium (Sambrook et al., 1989, Molecular Cloning, aLaboratory Manual, 2. edition, Cold Spring Harbor Laboratory Press)supplemented with 1 g/l casaminoacids, 500 micrograms/l thiamine and 10mg/l kanamycin.

Media and Substrates

Cove was composed of 342.3 g/L Sucrose, 20 ml/L COVE salt solution, 10mM Acetamide and 30 g/L noble agar. Cove-2 was composed of 30 g/LSucrose, 20 ml/L COVE salt solution, 10 mM, Acetamide and 30 g/L nobleagar. COVE salt solution was composed of 26 g/L KCl, 26 g/L MgSO₄.7H₂O,76 g/L KH₂PO₄ and 50 ml/L Cove trace metals. COVE trace metals wascomposed of 0.04 g/L Na₂B₄O₇.10H₂O, 0.4 g/L CuSO₄.5H₂O, 1.2 g/LFeSO₄.7H₂O, 1.0 g/L MnSO₄.H₂O, 0.8 g/L Na₂MoO₂.2H₂O and 10 g/LZnSO₄.7H₂O. YPG was composed of 4 g/L yeast extract, 1 g/L K₂HPO₄, 0.5g/L MgSO₄.7H₂O and 15 g/L glucose, pH 6.0. STC was composed of 0.8 MSorbitol, 25 mM Tris pH 8 and 25 mM CaCl₂. STPC was composed of 40%PEG4000 in STC buffer. Cove top agarose was composed of 342.3 g/LSucrose, 20 ml/L COVE salt solution, 10 mM Acetamide and 10 g/L low meltagarose. MLC was composed of 40 g/L Glucose, 50 g/L Soybean powder, 4g/L Citric acid, pH 5.0. G0-50 was composed of glucose 50 g/L, KH2PO4 2g/L, MgSO4-7aq 2 g/L, K2SO4 3 g, citric acid 3 g/L, oxalic acid 50 g/L,AMG trace metal solution 0.5 m/L and urea 3 g/L, pH 5.0. AMG trace metalsolution was composed of 6.8 g/L ZnCl₂.7H₂O, 2.5 g/L CuSO₄.5H₂O, 0.24g/L NiCl₂.6H₂O, 13.9 g/L FeSO₄.7H₂O, 13.5 g/L MnSO₄.H₂O and 3 g/L citricacid.

Acid Stable Alpha-Amylase Activity

When used according to the present invention the activity of any acidstable alpha-amylase may be measured in AFAU (Acid Fungal Alpha-amylaseUnits), which are determined relative to an enzyme standard. 1 FAU isdefined as the amount of enzyme which degrades 5.260 mg starch drymatter per hour under the below mentioned standard conditions.

Acid stable alpha-amylase, an endo-alpha-amylase(1,4-alpha-D-glucan-glucano-hydrolase, E.C. 3.2.1.1) hydrolyzesalpha-1,4-glucosidic bonds in the inner regions of the starch moleculeto form dextrins and oligosaccharides with different chain lengths. Theintensity of color formed with iodine is directly proportional to theconcentration of starch. Amylase activity is determined using reversecolorimetry as a reduction in the concentration of starch under thespecified analytical conditions.

$\begin{matrix}{{STARCH} + {IODINE}} \\{\lambda = {590\mspace{14mu} {nm}}}\end{matrix}\mspace{14mu} \underset{\underset{{40{^\circ}},{{pH}\; 2},5}{\rightarrow}}{{ALPHA}\text{-}{AMYLASE}}\mspace{14mu} \begin{matrix}{{DEXTRINS} +} \\{OLIGOSACCHARIDES}\end{matrix}$ blue/violet t = 23  sec .decoloration

Standard Conditions/Reaction Conditions:

Substrate: Soluble starch, approx. 0.17 g/L Buffer: Citrate, approx.0.03 M Iodine (I2): 0.03 g/L CaCl₂: 1.85 mM pH: 2.50 ± 0.05 Incubationtemperature: 40° C. Reaction time: 23 seconds Wavelength: 590 nm Enzymeconcentration: 0.025 AFAU/mL Enzyme working range: 0.01-0.04 AFAU/mL

A folder EB-SM-0259.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S, Denmark, which folderis hereby included by reference.

Glucoamylase Activity

Glucoamylase activity may be measured in AmyloGlucosidase Units (AGU).The AGU is defined as the amount of enzyme, which hydrolyzes 1 micromolemaltose per minute under the standard conditions 37° C., pH 4.3,substrate: maltose 23.2 mM, buffer: acetate 0.1 M, reaction time 5minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucosedehydrogenase reagent so that any alpha-D-glucose present is turned intobeta-D-glucose. Glucose dehydrogenase reacts specifically withbeta-D-glucose in the reaction mentioned above, forming NADH which isdetermined using a photometer at 340 nm as a measure of the originalglucose concentration.

Amg Incubation:

Substrate: maltose 23.2 mM Buffer: acetate 0.1 M pH: 4.30 ± 0.05Incubation temperature: 37° C. ± 1 Reaction time: 5 minutes Enzymeworking range: 0.5-4.0 AGU/mL

Color Reaction:

GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21 mM Buffer: phosphate 0.12 M;0.15 M NaCl pH: 7.60 ± 0.05 Incubation temperature: 37° C. ± 1 Reactiontime: 5 minutes Wavelength: 340 nm

A folder (EB-SM-0131.02/01) describing this analytical method in moredetail is available on request from Novozymes A/S, Denmark, which folderis hereby included by reference.

DNA Manipulations

Unless otherwise stated, DNA manipulations and transformations wereperformed using standard methods of molecular biology as described inSambrook et al., 1989, Molecular cloning: A laboratory manual, ColdSpring Harbor lab. Cold Spring Harbor, N.Y.; Ausubel, F. M. et al.(eds.) “Current Protocols in Molecular Biology”, John Wiley and Sons,1995; Harwood, C. R. and Cutting, S. M. (eds.)

Example 1 Cloning of Carbohydrate Binding Module (CBM) from AspergillusKawachii

In order to clone the carbohydrate binding module (CBM) with linker fromA. kawachii, the primers CBM1 (SEQ ID NO:1) and CBM2 (SEQ ID NO:2) weredesigned based on the nucleotide sequences of Aspergillus kawachii acidstable alpha-amylase in the EMBL database (EMBL:#AB008370). CBM1 andCBM2 comprise a BamHI site and a SalI site, respectively.

CBM1: 5′-gaagggatccgatttttactagtacatccaaagccaccac-3′ CBM2:5′-tttgtcgacctacctccacgtatcaaccaccgtctcc-3′

PCR reaction was carried out with the primers CBM1 and CBM2 usinggenomic DNA from A. kawachii (IF04308) as template. Reaction components(1 ng/microL of genomic DNA, 250 mM dNTP each, primer 250 nM each, 0.1U/microL in Taq polymerase in 1× buffer (Roche Diagnostics, Japan)) weremixed and submitted for PCR under the following conditions.

Step Temperature Time 1 94° C. 2 min 2 92° C. 1 min 3 55° C. 1 min 4 72°C. 1 min 5 72° C. 10 min  6  4° C. forever Steps 2 to 4 were repeated 30times.

A 0.4 kb fragment comprising the CBM with linker region was amplified.The amplified DNA was cut by Sal I and BamH I and ligated into pMT2188digested by Xho I and BamH Ito create pHUda381 having CBM with linkerregion from Aspergillus kawachii, Aspergillus niger neutral amylasepromoter, Aspergillus nidulans TPI leader sequences, Aspergillus nigerglucoamylase terminator and Aspergillus nidulans amdS gene as a marker.The pHUda381 was sequenced to confirm presence of correct CBM withlinker sequence. The CBM with linker sequence is shown in SEQ ID NO:5.

Example 2 Expression of the Hybrid Enzyme in Aspergillus niger

The production of the cDNA sequence of Aspergillus niger acid stablealpha-amylase gene and the cDNA clone of Aspergillus niger acid stablealpha-amylase are described in WO 89/01969 (example 1 and 3). A PCRreaction with cDNA clone of Aspergillus niger acid stable alpha-amylaseas template was performed using the primers (SEQ ID NO:3) and (SEQ IDNO:4) to introduce a BamHI site and a SpeI site, respectively.

(SEQ ID NO: 3) 5′-tttggatccaccatgagattatcgacttcgagtctcttc-3′(SEQ ID NO: 4) 5′-tttactagtagcagcagcagttgtggtcgtggttgttc-3′

Reaction components (1 ng/microL of template DNA, 250 mM dNTP each,primer 250 nM each, 0.1 U/microL in Taq polymerase in 1× buffer (RocheDiagnostics, Japan)) were mixed and submitted for PCR under thefollowing conditions.

Step Temperature Time 1 94° C. 2 min 2 92° C. 1 min 3 55° C. 1 min 4 72°C. 2 min 5 72° C. 10 min  6  4° C. forever Steps 2 to 4 were repeated 30times.

The 1.5 kb amplified DNA fragment was cut by Spe I and BamH I andligated into pHUda381 digested by Spe I and BamH I to create theexpression plasmid pHUda387 comprising Aspergillus niger acid stablealpha-amylase cDNA fused with CBM from Aspergillus kawachii. ThepHUda387 was sequenced to confirm that no changes had happen in theAspergillus niger acid stable alpha-amylase cDNA sequences. TheAspergillus niger acid stable alpha-amylase cDNA sequence is shown inSEQ ID NO:7.

The pHUda387 was transformed into Aspergillus niger MBin120. The hoststrain was propagated in 100 ml of non-selective YPG medium at 32° C.for 16 hrs on a rotary shaker at 120 rpm. Cells were collected byfiltering, washed with 0.6 M KCl and resuspended in 20 ml of 0.6 M KClcontaining a commercial beta-glucanase product (GLUCANEX™, NovozymesA/S) at final concentration of 600 microL/ml. The suspension wasincubated at 32° C. at 80 rpm until protoplasts were formed, then washedtwice with STC buffer. The protoplasts were counted with a hematometerand resuspended and adjusted in an 8:2:0.1 solution of STC:STPC:DMSO toa final concentration of 2.5×10⁷ protoplasts/ml. About 3 micrograms ofplasmid DNA was added to 100 microL of protoplast suspension, mixedgently and incubated on ice for 20 min. One ml of SPTC was added and theprotoplast suspension was incubated for 30 min at 37° C. After theaddition of 10 ml of 50° C. Cove top agarose, the reaction was pouredonto Cove agar plates and the plates were incubated at 32° C. After 5days transformants were selected from the Cove medium.

The selected transformants were inoculated in 100 ml of MLC media andcultivated at 30° C. for 2 days. 10 ml of MLC medium was inoculated to100 ml of G0-50 medium and cultivated at 30° C. for 5 days. Thesupernatant was obtained by centrifugation.

The acid stable alpha-amylase activity in the supernatant was determinedas decrease of blue color of starch-iodine complex measured in OD 590nm. 25 microL of enzyme samples dissolved in sample buffer (51.4 mMcalcium chloride in 2 mM citrate buffer [pH 2.5]) was mixed with 135microL of substrate solution (0.6 g/L of starch [Merck 1253] and 12 g/Lof sodium acetate in 100 mM citrate buffer [pH 2.5]) and incubated at37° C. for 325 sec. After 325 sec, 90 microL of iodine solution (1.2 g/Lof potassium iodine [Merck 5043] and 0.12 g/L of iodine [Merck 4761])was added to the reaction mixture and incubated at 37° C. for 25 sec.Activity was measured at 590 nm on a spectrophotometer. 25 microL ofdistilled water was used instead of enzyme samples in blank experiments.

Example 3 Performance of the Hybrid Enzyme in SSF of Non-GelatinizedStarch

The relative performance of Aspergillus niger acid stable alpha-amylaseand Aspergillus niger acid stable alpha-amylase with an attachedcarbohydrate-binding module was evaluated via mini-scale SSF(Simultaneous Saccharification and Fermentation) fermentations. Dosagesused were 0.3, 0.5 and 1.0 AFAU/g DS of the respective alpha-amylasecomplemented with a 0.5 AGU/g DS dose of purified Aspergillus nigerglucoamylase. Briefly, approximately 1.9 g of ground corn (yellow #2dent corn ground in a pilot scale hammer mill and passed through a 2 mmscreen, 11.2% moisture content) was added to 16 ml polystyrene tubes(Falcon 352025). A solution of 0.02 N H₂SO₄ with 3 mg/ml penicillin wasadded in an amount appropriate to bring the dry solids level (DS) to 34%and the pH to 5.0. Treatments were made in six replicates.

After dosing the enzymes, the tubes were inoculated with 4.74×10⁸ yeastcells/ml. Tubes were capped with a screw on lid which had been puncturedwith a very small needle to allow gas release and vortexed brieflybefore weighing and incubation at 32° C. Fermentation progress wasfollowed by weighing the tubes over time. Tubes were vortexed brieflybefore weighing. The relationship used between amount of CO₂ loss andthe weight of ethanol was: CO₂ loss (g)×1.045=EtOH (g). The results areshown in table 2.

TABLE 2 Average ethanol yield as g ethanol/g DS after 50 hrs.Aspergillus niger Aspergillus niger Dose (AFAU/g DS) alpha-amylasealpha-amylase + CBM 0.3 0.342 0.366 0.5 0.348 0.383 1.0 0.365 0.390 95%confidence limits: +/−0.008

On an equal activity basis, Aspergillus niger acid stable alpha-amylasewith an attached carbohydrate-binding module (hybrid) significantlyoutperformed Aspergillus niger acid stable alpha-amylase (native) at alldosages tested. The hybrid enzyme was approximately three times moreeffective than the native enzyme (i.e., performance of Aspergillus nigeracid stable alpha-amylase could be matched with about three times lessAspergillus niger acid stable alpha-amylase with an attachedcarbohydrate-binding module).

Example 4 Performance of Hybrid Enzymes with Different Combinations ofCBM, Linker Sequence and Catalytic Module

Hybrid enzyme variants comprising the Aspergillus niger acid stablealpha-amylase (AA) and a CBM from A. kawachii alpha-amylase, A. nigerAMG, T. emersonii AMG, Athelia rolfsii AMG, or Bacillus sp. maltogenicalpha-amylase (Novamyl) were constructed. The linker sequence from A.kawachii alpha-amylase (SEQ ID NO:27) was used in all the variants.

The performance of the variants was assessed using corn starch (SIGMAS-9679) as substrate, Aspergillus niger glucoamylase G2 0.5 AGU/g DS,variant 1.0 AFAU/g DS and quantification of the liberated glucose withthe Glucose B-test kit (Wako Pure Chemicals 271-3141). The results areshown in table 3.

TABLE 3 Performance of hybrid enzymes with different CBMs. Glucose mg/mlafter 20, 48, and 68 hrs (1.0 AFAU/gDS) Variant Catalytic module LinkerSBD 20 hrs 48 hrs 68 hrs JA001 Aspergillus niger kawachii AA kawachii AA0.18 0.51 1.07 JA002 Aspergillus niger kawachii AA niger AMG 0.38 1.081.88 JA003 Aspergillus niger kawachii AA emersonii AMG 0.04 0.19 0.43JA004 Aspergillus niger kawachii AA rolfsii AMG 0.48 1.43 2.48 JA005Aspergillus niger kawachii AA Bacillus MA −0.03 −0.04 0.10

Hybrid enzyme variants comprising the Aspergillus niger acid stablealpha-amylase and a CBM from A. kawachii alpha-amylase, A. niger AMG, orAthelia rolfsii AMG, were constructed. Also variants with differentlinker sequence were constructed. The linker sequences used were A.niger AMG linker (SEQ ID NO:26), A. kawachii alpha-amylase linker (SEQID NO:27), Athelia rolfsii AMG linker (SEQ ID NO:28), and the PEPTlinker (SEQ ID NO:29). A variant having two CBDs in tandem was alsoconstructed (JA012).

The performance of the variants was assessed as above, only withAspergillus niger glucoamylase G1 and the variant was dosed as 0.3AFAU/g DS. The results are shown in table 4.

TABLE 4 Performance of hybrid enzymes with different linker sequencesand catalytic modules. Glucose mg/ml after 18, 24, and 42 hrs. VariantCatalytic module Linker SBD 18 hrs 24 hrs 42 hrs 68 hrs JA001Aspergillus niger kawachii AA kawachii AA 2.73 4.40 7.01 8.93 JA002Aspergillus niger kawachii AA niger AMG 2.75 4.30 7.03 9.23 JA004Aspergillus niger kawachii AA rolfsii AMG 2.91 4.35 7.16 9.11 JA008Aspergillus niger niger AMG niger AMG 3.01 4.26 7.31 8.76 JA009Aspergillus niger rolfsii AMG niger AMG 3.02 4.69 7.60 9.11 JA010Aspergillus niger PEPT niger AMG 3.18 4.66 7.82 9.38 JA011 Aspergillusniger rolfsii AMG rolfsii AMG 3.31 4.62 7.49 9.60 JA012 Aspergillusniger kawachii AA niger AMG + 2.99 4.10 6.97 8.85 rolfsii AMG

Hybrid enzyme variants comprising the linker and CBM from A. kawachiialpha-amylase with different catalytic modules were constructed. Thecatalytic modules applied were from Aspergillus niger acid stablealpha-amylase, A. kawachii alpha-amylase or Aspergillus oryzaealpha-amylase (Fungamyl™). The performance of the variants was assessedas above, with Aspergillus niger glucoamylase G1 and the variant dosedas 0.3 AFAU/g DS. The results are shown in table 5.

TABLE 5 Performance of hybrid enzymes with different catalytic modules.Glucose mg/ml after 18, 24, 42 and 68 hrs. Variant catalytic moduleLinker SBD 18 hrs 24 hrs 42 hrs 68 hrs JA001 A. niger AA kawachii AAkawachii AA 2.73 4.40 7.01 8.93 JA006 A. oryzae AA kawachii AA kawachiiAA 3.39 5.10 7.99 9.77 JA007 A. kawachii AA kawachii AA kawachii AA 2.913.99 6.82 8.57

Hybrid enzyme variants comprising the linker and CBM from A. kawachiialpha-amylase or A. rolfsii AMG with different catalytic modules fromAspergillus niger acid stable alpha-amylase or Aspergillus oryzaealpha-amylase (Fungamyl™). The performance of the variants was assessedas above with Aspergillus niger glucoamylase G1 and the variant dosed as0.3 AFAU/g DS. The results are shown in table 6.

TABLE 6 Performance of different hybrid enzymes. Glucose mg/ml after 18,24 or 42 hrs. Variant catalytic module Linker SBD 18 hrs 24 hrs 42 hrsJA001 A. niger AA kawachii AA kawachii AA 4.64 5.61 8.38 JA006 A. oryzaeAA kawachii AA kawachii AA 6.27 7.93 9.40 JA017 A. oryzae AA rolfsii AMGrolfsii AMG 6.68 8.87 9.80

Example 5 Variants of JA017 with One or More Substitutions in theCatalytic Module

Variants of the hybrid enzyme JA017 (amino acid sequence shown in SEQ IDNO:40) comprising the A. rolfsii AMG CBM, A. rolfsii linker sequence andthe A. oryzae alpha amylase catalytic module with one or moresubstitutions in the catalytic module were constructed usingconventional protein engineering techniques. Table 7 lists thesubstitutions in the variants.

TABLE 7 Variants of JA017 (SEQ ID NO: 40) with one or more substitutionsin the catalytic module. No. Substitution JA019 Y175W E176D JA050 N264KM266L JA056 D253N JA057 K158D S161D Q163S D164S G466D D468S N470D JA059G60N N264K M266L JA060 D177N JA061 K158D S161D Q163S D164S Y175W E176DG466D D468S N470D JA062 K158D S161D Q163S D164S Y175W E176D N264K M266LG466D D468S N470D JA063 K158V S161D Q163S D164S G466D D468S N470D JA069K158D S161N Q163A D164S G466D D468S N470D JA074 K158V S161N Q163A D164SN264K M266L G466D D468S N470D JA076 Q81R K158V S161N Q163A D164S Y175WE176D G466D D468S N470D JA083 K158D S161D Q163S D164S Y175W E176D N264EM266L G466D D468S N470D JA085 Q81R K158V S161N Q163A D164S Y175W E176DD177N N264K M266L G466D JA093 Q81R K158V S161N Q163A D164S Y175W E176DD177N N264E M266L G466D JA094 K158V S161N Q163A D164S Y175W E176D D177NN264K M266L G466D D468S JA095 K158V S161N Q163A D164S Y175W E176D D177NN264E M266L G466D D468S JA096 K158V S161N Q163A D164S D177N N264K M266LG466D D468S N470D JA097 K158V S161N Q163A D164S D177N N264E M266L G466DD468S N470D

The variants were expressed in Aspergillus oryzae and characterized forpH stability, thermostability and the performance toward raw starch byglucose releasing test.

JA085 showed at least by 5° C. higher thermostability than JA017 at pH4.5 was and stable even at pH 3.0.

The new variants JA085 and JA074 showed a high specific activity in theglucose release test. JA085 continued to release glucose even at pH 3.8where JA017 was quickly inactivated. JA074 had the highest glucoseyields at pH 4.0 was but was inactivated at pH 3.8.

Tables 8-10 list the temperature stability as residual activity after 30min incubation at 55° C. and 60° C. at pH 4.5, the specific activity,the pH stability and the activity in the glucose releasing test of anumber of variants.

TABLE 8 Temperature stability and specific activity of selected PEvariants Temperature stability (%) in 1 mM CaCl₂ No CaCl₂ Specificactivity 55° C. 60° C. 55° C. (mFAU/OD280) JA017 2 0 0 3157 JA057 12 0 0JA061 51 0 16 JA063 18 0 0 JA069 16 0 0 JA074 19 0 0 JA085 68 20 12 3653JA095 59 7 2 4240 JA096 53 0 0 JA097 40 0 0

Table 9a. Temperature stability (pH4.5, 1 hour incubation) Temp. °JA017A JA085A 35 101 107 40 109 102 45 98 96 50 64 95 55 0 56 60 0 27 650 0 Table 9b. pH stability (32° C., 1 hour incubation) pH JA017A JA085A2.5 0 20 3.0 23 76 3.5 74 83 4.0 88 92 4.5 86 95 5.0 96 99 5.5 100 100

0 min 19 min 43 min 67 min 91 min Table 10a. Performance in glucosereleasing test with purified A. niger AMG G2 at pH 4.0. Glucose mg/ml.JA001 0.48 3.48 5.67 8.26 10.79 JA017 0.46 6.45 7.97 8.71 8.98 JA0740.44 8.85 14.69 18.24 20.29 JA085 0.41 5.87 10.62 13.92 17.34 Table 10b.Performance in glucose releasing test with purified A. niger AMG G2 atpH 3.8. Glucose mg/ml. JA001 0.48 3.19 5.28 7.63 10.32 JA017 0.46 4.284.71 4.98 5.13 JA074 0.43 7.75 11.58 13.55 13.93 JA085 0.41 5.71 9.7312.99 15.76

0 min 18 min 90 min Table 11a. Performance in glucose releasing testwith purified A. niger AMG G2 at pH 3.9. Glucose mg/ml. JA074 0.41 6.8316.87 JA085 0.40 4.50 14.08 JA094 0.40 4.48 14.00 JA095 0.40 4.49 13.82Table 11b. Performance in glucose releasing test with purified A. nigerAMG G2 at pH 3.6. Glucose mg/ml. JA074 0.43 5.01 7.57 JA085 0.43 4.1810.61 JA094 0.42 4.18 11.18 JA095 0.42 4.33 11.52

Example 6 Variants of the Aspergillus kawachii Acid Alpha-Amylase

A variant of the Aspergillus kawachii acid alpha-amylase suitable forraw starch hydrolysis may be produced by conventional protein engineeredof the sequence comprising the catalytic module. Specific positionwherein alterations can improve specific activity and/or stability waspredicted based on similarity to one of the following fungalalpha-amylases having the indicated amino acid sequence and athree-dimensional structure found under the indicated identifier in theRCSB Protein Data Bank (www.rcsb.org): the A. niger acid alpha-amylase(2aaa, SEQ ID NO:42) and the alpha-amylase (TAKA amylase) fromAspergillus oryzae (6taa or 7taa, SEQ ID NO:43). The two 3D models weresuperimposed by aligning the amino acid residues of each catalytictriad. This was done by methods known in the art based on the deviationsof the three pairs of C-alpha atoms, e.g., by minimizing the sum ofsquares of the three deviations or by aligning so as to keep eachdeviation below 0.8 Å, e.g., below 0.6 Å, below 0.4 Å, below 0.3 Å orbelow 0.2 Å.

Alternatively, the superimposition may be done by aligning the two aminoacid sequences by a conventional method and minimizing the sum ofsquares of the deviations of all corresponding pairs of amino acidresidues. The sequence alignment may be done by conventional methods,e.g., by use the software GAP from UWGCG Version 8.

On the alignment of the three alpha-amylases from A. kawachii, A. niger(aaa_new) and A. oryzae (7taa) shown below are indicated the residueswhich are within 10 Å from the acarbose substrate in 7taa.pdb structure.These residues are targets for alterations conferring improved specificactivity.

kawachi MRVSTSSIAL AVSLFGKLAL GLSAAEWRTQ SIYFLLTDRF GRTDNSTTAT 50aaa_new MRLSTSSLFL SVSLLGKLAL GLSAAEWRTQ SIYFLLTDRF GRTDNSTTAT 7taa.......... .......... .ATPADWRSQ SIYFLLTDRF ARTDGSTTAT 29                                    * *  * kawachiCNTGDQIYCG GSWQGIINHL DYIQGMGFTA IWISPITEQL PQDTSDGEAY 100 aaa_newCDTGDQIYCG GSWQGIINHL DYIQGMGFTA IWISPITEQL PQDTADGEAY 7taaCNTADQKYCG GTWQGIIDKL DYIQGMGFTA IWITPVTAQL PQTTAYGDAY 79  *****                           * **   **    ******* kawachiHGYWQQKIYY VNSNFGTADD LKSLSDALHA RGMYLMVDVV PNHMGYAGNG 150 aaa_newHGYWQQKIYD VNSNFGTADD LKSLSDALHA RGMYLMVDVV PNHMGYAGNG 7taaHGYWQQDIYS LNENYGTADD LKALSSALHE RGMYLMVDVV ANHMGYDGAG 129*****                                   *** ****** kawachiNDVDYSVFDP FDSSSYFHPY CLITDWDNLT MVQDCWEGDT IVSLPDLNTT 200 aaa_newNDVDYSVFDP FDSSSYFHPY CLITDWDNLT MVQDCWEGDT IVSLPDLNTT 7taaSSVDYSVFKP FSSQDYFHPF CFIQNYEDQT QVEDCWLGDN TVSLPDLDTT 179                        *******   **  ***** ****** kawachiETAVRTIWYD WVADLVSNYS VDGLRIDSVE EVEPDFFPGY QEAAGVYCVG 250 aaa_newETAVRTIWYD WVADLVSNYS VDGLRIDSVL EVEPDFFPGY QEAAGVYCVG 7taaKDVVKNEWYD WVGSLVSNYS IDGLRIDTVK HVQKDFWPGY NKAAGVYCIG 229                          ****** **    *             * kawachiEVDNGNPALD CPYQKYLDGV LNYPIYWQLL YAFESSSGSI SNLYNMIKSV 300 aaa_newEVDNGNPALD CPYQKVLDGV LNYPIYWQLL YAFESSSGSI SNLYNMIKSV 7taaEVLDGDPAYT CPYQNVMDGV LNYPIYYPLL NAFKSTSGSM DDLYNMINTV 279**********   *        * ** *** * *               * kawachiASDCSDPTLL GNFIENHDNP RFASYTSDYS QAKNVLSYIF LSDGIPIVYA 350 aaa_newASDCSDPTLL GNFIENHDNP RFASYTSDYS QAKNVLSYIF LSDGIPIVYA 7taaKSDCPDSTLL GTFVENHDNP RFASYTNDIA LAKNVAAFII LNDGIPIIYA 329             *  *****    *                          * kawachiGEEQHYSGGD VPYNREATWL SGYDTSAELY TWIATTNAIR KLAISADSDY 400 aaa_newGEEQHYSGGK VPYNREATWL SGYDTSAELY TWIATTNAIR KLAISADSAY 7taaGQEQHYAGGN DPANREATWL SGYPTDSELY KLIASANAIR NYAISKDTGF 379         * ***** kawachiITYKNDPIYT DSNTIAMRKG TSGSQIITVL SNKGSSGSSY TLTLSGSGYT 450 aaa_newITYANDAFYT DSNTIAMRKG TSGSQVITVL SNKGSSGSSY TLTLSGSGYT 7taaVTYKNWPIYK DDTTIAMRKG TDGSQIVTIL SNKGASGDSY TLSLSGAGYT 429 kawachiSGTKLIEAYT CTSVTVDSNG DIPVPMASGL PRVLLPASVV DSSSLCGGSG 500 aaa_newSGTKLIEAYT CTSVTVDSSG DIPVPMASGL PRVLLPASVV DSSSLCGGSG 7taaAGQQLTEVIG CTTVTVGSDG NVPVPMAGGL PRVLYPTEKL AGSKICS... 474 kawachiNTTTTTTAAT STSKATTSSS SSSAAATTSS SCTATSTTLP ITFEELVTTT 550 aaa_newRLYVE..... .......... .......... .......... .......... 505 7taa.......... .......... .......... .......... .......... kawachiYGEEVYLSGS ISQLGEWHTS DAVKLSADDY TSSNPEWSVT VSLPVGTTFE 600 aaa_new.......... .......... .......... .......... .......... 7taa.......... .......... .......... .......... ..........601                                     640 kawachiYKFIKVDEGG SVTWESDPNR EYTVPECGSG SGETVVDTWR 640 aaa_new.......... .......... .......... .......... 7taa.......... .......... .......... ..........

The specific alterations for increased specific activity is onesubstitution or a combination of substitutions in the followingpositions within the “10 Å distance of the substrate”: 13, 15, 18, 32-36(i.e., 32, 33, 34, 35, 36), 61, 63-64, 68-69, 73-84 (e.g., 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84), 117-125 (e.g., 117, 118, 119, 120,121, 122, 123, 124, 125), 152-158 (e.g., 152, 153, 154, 155, 156, 157,158), 161-162, 165-175 (e.g., 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175), 204-211 (e.g., 204, 205, 206, 207, 208, 209, 210, 211),216, 229-239 (229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239),242, 250, 252-253, 255-257 (e.g., 255, 256, 257), 259-260, 275, 292,295-299 (e.g., 295, 296, 297, 298, 299), 304, 328, 339-344 according tothe 7taa numbering in the alignment above where the N-terminal is ATPADin 7taa (TAKA amylase). Most interesting positions are: 33, 36, 74, 75,77, 120, 153, 154, 155, 156, 157, 158, 162, 166, 169, 170, 199, 232,233, 235, 238, 239, 256, 257, 331, 336, 339, 340, and 342. Thesubstitutions may be to any amino acid residue.

Variants comprising one of the following substitutions or combinationsthereof may be produced: G33A, 136K, S74A, D75Y, E77D, P120A, I153D,D154N, W155Y, D156E, N157D, L158Q, Q162E, E166L, T169N, I170T, E199K,E199L, D232L, N233D, N235D, L238Y, D239T, W256Y, Q257P, E331Q, S336A,D339K, D339N, V340D, and Y342A. The most interesting variants compriseone or more of the following substitutions; S74A, E166L, E199L, D339K,and D156E. Yet more interesting is the variant having the multiplesubstitutions S74A/E166L/E199L.

Based on similarity between Aspergillus kawachii and the A. niger acidalpha-amylases the following substitutions is predicted to improve thespecific activity; N31D, S74A, Y89D, E209L, Y245V, D348K, D378A, K383A,P386A, I387F, I405V, N448S, and N480R, and a variant named WA01comprising these substitutions can be produced.

Example 7 Performance of a Variant of the Aspergillus kawachii AcidAlpha-Amylase

The sequences of the hybrids JA007 and JA001 described in example 4 areidentical to respectively the sequence of the A. kawachii acidalpha-amylase (SEQ ID NO:41) and the sequence of the variant WA01described in the previous example. As the two hybrids JA001 and JA007have been compared in several tests the improvement in specific activityconferred by the substitutions to the variant WA01 can be veryaccurately predicted. Specific activity of the variant WA01 will be 1.7to 1.8 times higher than the specific activity of the A. kawachii wildtype amylase. The prediction is based on the data shown in table 12.

TABLE 12 Performance of the hybrid JA007 (identical to A. kawachii acidalpha-amylase) and the hybrid JA001 (identical to WA01, a variant of theA. kawachii acid alpha-amylase comprising the substitutions N31D, S74A,Y89D, E209L, Y245V, D348K, D378A, K383A, P386A, I387F, I405V, N448S, andN480R) AFAU/ml mg/ml AFAU/mg A280 AFAU/A280 JA001 1.41 0.67 2.11 1.221.15 JA007 0.56 0.46 1.22 0.89 0.63

Example 8 Raw Starch Hydrolysis with an Acid Fungal Alpha-Amylase withor without CBD

This example illustrates the conversion of granular wheat starch or corngrits into glucose using acid fungal amylase without a CBM (SEQ ID NO:8)or the corresponding hybrid with a CBM (JA001). A slurry with 33% drysolids (DS) granular starch was prepared by adding 247.5 g of wheatstarch or maize grits under stirring to 502.5 ml of water. The pH wasadjusted with HCl to 4.5. The granular starch slurry was distributed to100 ml blue cap flasks with 75 g in each flask. The flasks wereincubated with magnetic stirring in a 60° C. water bath. At zero hoursthe enzyme activities were dosed to the flasks; glucoamylase (200 AGU/kgDS), an acid fungal amylase (50 AFAU/kg DS) and the wild typeAspergillus niger acid alpha-amylase (SEQ ID NO:8) or the Aspergillusniger acid alpha-amylase but fused to a linker and CBM derived from A.kawachii (SEQ ID NO:8 fused to SEQ ID NO:6). The Aspergillus niger acidalpha-amylase w/wo CBM was dosed as 100 KNU/kg DS. Samples werewithdrawn after 24, 48, 72, and 96 hours.

Total dry solids starch was determined using the following method. Thestarch was completely hydrolyzed by adding an excess amount ofalpha-amylase (300 KNU/kg dry solids) and placing the sample in an oilbath at 95° C. for 45 minutes. Subsequently the samples were cooled to60° C. and an excess amount of glucoamylase (600 AGU/kg DS) was addedfollowed by incubation for 2 hours at 60° C.

Soluble dry solids in the starch hydrolysate were determined byrefractive index measurement on samples after filtering through a 0.22microM filter. The sugar profiles were determined by HPLC. The amount ofglucose was calculated as DX.

The results are shown in tables 13-14 (wheat starch) and 15-16 (corngrits).

TABLE 13 Soluble dry solids as percentage of total dry substance fromwheat starch. Enzymes: glucoamylase, acid fungal alpha-amylase and moreacid fungal alpha-amylase with the CBM or without the CBM. 24 hours 46hours 70 hours 90 hours Without CBM 81.1 88.6 89.4 90.7 With CBM 86.292.6 93.7 95.1

TABLE 14 The DX of the soluble hydrolysate from wheat starch: Enzymes:glucoamylase, acid fungal amylase and more acid fungal alpha-amylasewith the CBM or without the CBM. 24 hours 46 hours 70 hours 90 hoursWithout CBM 78.1 84.9 85.6 86.7 With CBM 82.3 88.4 89.5 905

TABLE 15 Soluble dry solids as percentage of total dry substance fromcorn grits. Enzymes: glucoamylase, acid fungal alpha-amylase and moreacid fungal alpha-amylase with the CBM or without the CBM. 24 hours 46hours 70 hours 90 hours Without CBM 44.0 49.6 56.8 62.2 With CBM 53.659.7 63.8 68.4

TABLE 16 The DX of the soluble hydrolysate from corn grits: Enzymes:glucoamylase, acid fungal amylase and more acid fungal alpha-amylasewith the CBM or without the CBM. 24 hours 46 hours 70 hours 90 hoursWithout CBM 42.1 47.3 53.9 58.8 With CBM 51.2 56.8 60.4 64.5

1. A hybrid enzyme which comprises an amino acid sequence of a catalyticmodule and an amino acid sequence of a carbohydrate binding module,wherein (a) the catalytic module is an amino acid sequence which has atleast 70% homology to the amino acid sequence of SEQ ID NO:8 or an aminoacid sequence which has at least 70% homology to the amino acid sequenceof SEQ ID NO:30; and (b) the carbohydrate binding module is an aminoacid sequence having at least 50% homology to an amino acid sequenceselected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25.
 2. Thehybrid enzyme of claim 1, wherein the hybrid enzyme further comprises alinker amino acid sequence which connects the amino acid sequence of thecatalytic module and the amino acid sequence of the carbohydrate-bindingmodule.
 3. The hybrid enzyme of claim 2, wherein the linker sequence isa linker sequence selected from the group consisting of the linkersequence of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29or a linker sequence which differs from the amino acid sequence shown inSEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29 by no morethan 10 positions, no more than 9 positions, no more than 8 positions,no more than 7 positions, no more than 6 positions, no more than 5positions, no more than 4 positions, no more than 3 positions, no morethan 2 positions, or no more than 1 position.
 4. The hybrid enzyme ofclaim 1, wherein the catalytic module is an amino acid sequence whichhas at least 70% homology to the amino acid sequence of SEQ ID NO:8. 5.The hybrid enzyme of claim 1, wherein the catalytic module is an aminoacid sequence which has at least 80% homology to the amino acid sequenceof SEQ ID NO:8.
 6. The hybrid enzyme of claim 1, wherein the catalyticmodule is an amino acid sequence which has at least 90% homology to theamino acid sequence of SEQ ID NO:8.
 7. The hybrid enzyme of claim 1,wherein the catalytic module is an amino acid sequence which has atleast 95% homology to the amino acid sequence of SEQ ID NO:8.
 8. Thehybrid enzyme of claim 1, wherein the catalytic module is an amino acidsequence which has at least 70% homology to the amino acid sequence ofSEQ ID NO:30.
 9. The hybrid enzyme of claim 1, wherein the catalyticmodule is an amino acid sequence which has at least 80% homology to theamino acid sequence of SEQ ID NO:30.
 10. The hybrid enzyme of claim 1,wherein the catalytic module is an amino acid sequence which has atleast 90% homology to the amino acid sequence of SEQ ID NO:30.
 11. Thehybrid enzyme of claim 1, wherein the catalytic module is an amino acidsequence which has at least 95% homology to the amino acid sequence ofSEQ ID NO:30.
 12. The hybrid enzyme of claim 1, wherein the carbohydratebinding module is an amino acid sequence having at least 60% homology toany of the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25.
 13. Thehybrid enzyme of claim 1, wherein the carbohydrate binding module is anamino acid sequence having at least 70% homology to any of the aminoacid sequences shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25.
 14. The hybrid enzymeof claim 1, wherein the carbohydrate binding module is an amino acidsequence having at least 80% homology to any of the amino acid sequencesshown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24 or SEQ ID NO:25.
 15. The hybrid enzyme of claim 1,wherein the carbohydrate binding module is an amino acid sequence havingat least 90% homology to any of the amino acid sequences shown in SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24 or SEQ ID NO:25.
 16. A hybrid enzyme comprising the catalyticdomain of the A. oryzae alpha amylase shown as amino acids 21-498 of thesequence SEQ ID NO:40 and an amino acid sequence having at least 70%homology to the sequence of amino acids 21-498 of SEQ ID NO:40.
 17. Avariant alpha-amylase having at least 80% homology to SEQ ID NO:41,which variant comprises an alteration at one or more of the positionsselected from the group consisting of: 13, 15, 18, 31, 32, 33, 34, 35,36, 61, 63, 64, 68, 69, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,89, 117, 118, 119, 120, 121, 122, 123, 124, 125, 152, 153, 154, 155,156, 157, 158, 161, 162, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 204, 205, 206, 207, 208, 209, 210, 211, 216, 229, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 242, 245, 250, 252, 253, 255,256, 257, 259, 260, 275, 292, 295, 296, 297, 298, 299, 304, 328, 339,344, 348, 378, 383, 386, 387, 405, 448 and 480, wherein (a) thealteration(s) are independently, (i) an insertion of an amino aciddownstream of the amino acid which occupies the position, (ii) adeletion of the amino acid which occupies the position, or (iii) asubstitution of the amino acid which occupies the position with adifferent amino acid, (b) the variant has alpha-amylase activity. 18-21.(canceled)
 22. An isolated DNA sequence encoding the hybrid enzyme ofclaim
 1. 23-26. (canceled)
 27. A method for liquefying starch,comprising treating a gelatinized or granular starch substrate with thehybrid enzyme of claim 1 in an aqueous medium. 28-30. (canceled)
 31. Aprocess for preparing a dough-based product, comprising adding thehybrid enzyme of claim 1 to a dough. 32-36. (canceled)